Poliovirus receptor (PVR/CD155) knockout cells derived from RD (human rhabdomyosarcoma) cell line by CRISPR

Abstract

A modified polio virus receptor (PVR/CD155) gene including one or more mutations in exons selected from exons 2, 3 and 4 of poliovirus receptor (PVR/CD155) gene having SEQ ID No.1. More specifically, cell lines including the modified gene and method of producing the same using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9) system. The cell line is refractory (non-permissive) to poliovirus and susceptible to most Enteroviruses and many other human viruses. Further, the cell line can be applied in the fields of research, diagnostic and therapy.

The invention claimed is: 
     
         1 . A modified poliovirus receptor (PVR/CD 155) gene comprising a sequence selected from SEQ ID Nos. 2-10. 
     
     
         2 . A genetically engineered cell strain comprising the modified poliovirus receptor (PVR/CD 155) gene as claimed in  claim 1 , wherein the genetically engineered cell strain is selected from the group consisting of RD-SJ1, RD-SJ4, RDSJ15, RD-SJ23, RD-SJ 28, RD-SJ30, RD-SJ35, RD- SJ37 and RD-SJ40, wherein the genetically engineered cell strain is derived from RD (Rhabdomyosarcoma) cell line of human rhabdomyosarcoma. 
     
     
         3 . The genetically engineered cell strain as claimed in  claim 2 , wherein the genetically engineered cell strain is refractory (non-permissive) to poliovirus and susceptible to enteroviruses and human viruses. 
     
     
         4 . A method for producing the genetically engineered cell strain as claimed in  claim 2 , the method comprising:
 modifying one or more target exons of a gene in the cell by introducing three or more guide RNAs selected from SEQ ID Nos. 19-23 and their oligonucleotides having sequences set forth in SEQ ID Nos. 24-33 for the target gene using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9) system;   knocking out one or more target exons of the gene by (i) causing five kinds of the three or more guide RNAs to target each of the one or more kinds of-target exons of the gene and then (ii) causing a Cas protein to cut each of the one or more kinds of target exons of the gene; and   sequencing the cell strain to confirm the knock-out using primers.   
     
     
         5 . The method as claimed in  claim 4 , wherein the primers have sequences set forth in SEQ ID Nos. 34-47. 
     
     
         6 . The method as claimed in  claim 4 , wherein the CRISPR-Cas9 system is a system including three or more guide RNAs for each of the one or more target genes. 
     
     
         7 . The method as claimed in  claim 4 , wherein the target exons of gene have sequence selected from SEQ ID Nos. 11-18. 
     
     
         8 . The method as claimed in  claim 4 , wherein the modified cell strain is a RD cell strain of human rhabdomyosarcoma. 
     
     
         9 . A method comprising applying the modified poliovirus receptor (PVR/CD 155) gene as claimed in  claim 1  for research. 
     
     
         10 . The genetically engineered cell strain as claimed in  claim 2 , wherein the genetically engineered cell strain comprises one or more target exons modified by introducing three or more kinds of guide RNAs selected from the group consisting of SEQ ID-NO Nos. 19-23. 
     
     
         11 . The genetically engineered cell strain as claimed in  claim 2 , wherein the genetically engineered cell strain is a knock-out cell line, and wherein primer sequences used for sequencing knock-out cell line are selected from the group consisting of SEQ ID Nos. 34-47.